This invention relates to delivery of chemical agents to cells. More particularly, this invention relates to compositions and methods for intracellular delivery of chemical agents to a specific cell type, i.e. cells bearing the interleukin-2 (IL-2) receptor.
Toxins that target cell surface receptors or antigens on tumor cells have attracted considerable attention for treatment of cancer. E.g., I. Pastan and D. FitzGerald, Recombinant Toxins for Cancer Treatment, 254 Science 1173 (1991); Anderson et al., U.S. Pat. Nos. 5,169,933 and 5,135,736; Thorpe et al., U.S. Pat. No. 5,165,923; Jansen et al., U.S. Pat. No. 4,906,469; Frankel, U.S. Pat. No. 4,962,188; Uhr et al., U.S. Pat. No. 4,792,447; Masuho et al., U.S. Pat. Nos. 4,450,154 and 4,350,626. These agents include a cell-targeting moiety, such as a growth factor or an antigen-binding protein, linked to a plant or bacterial toxin. They kill cells by mechanisms different from conventional chemotherapy, thus potentially reducing or eliminating cross resistance to conventional chemotherapeutic agents.
Copending U.S. patent application Ser. No. 08/305,770, filed Sep. 13, 1994, describes compositions and methods for specific intracellular delivery of a chemical agent into a CR2-receptor-bearing cell, e.g. B lymphocytes. The compositions comprise a CR2-receptor-binding and endocytosis-inducing ligand (CBEL) coupled to the chemical agent. The CBEL binds to the CR2 receptor on the surface of B lymphocytes and elicits endocytosis of the composition such that the composition is transported to lysosomes. In the lysosomes, the chemical agent is preferably separated from the remainder of the composition such that the chemical agent can be transported or diffuse into the cytoplasm or nucleus. Optionally, the composition can include a spacer, which can be either biodegradable (in the lysosome) or non-biodegradable, for coupling the CBEL to the chemical agent. Chemical agents can include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like. The composition can further comprise a carrier such as another water soluble polymer, liposome, or particulate.
Copending U.S. patent application Ser. No. 08/616,693, filed Mar. 15, 1996, and Ser. No. 08/857,009, filed May 15, 1997, describe compositions and methods for specific intracellular delivery of a chemical agent into T lymphocytes. The compositions are represented by the formula [Lxe2x80x94S]axe2x80x94Cxe2x80x94[Sxe2x80x94A]b wherein L is a ligand configured for binding to a receptor on a T lymphocyte and stimulating receptor-mediated endocytosis of the composition, A is a chemical agent, S is a spacer moiety, C is a water soluble polymer having functional groups compatible with forming covalent bonds with the ligand, chemical agent, and spacer, and a and b are positive integers. These compositions are also designed to be transported to lysosomes, where the chemical agent is separated from the remainder of the composition for diffusion or transport to other locations in the cell. Preferred water soluble polymers include poly(ethylene glycol) and a copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA). Preferred chemical agents include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like. The composition can further comprise a carrier such as other water soluble polymers, liposomes, or particulates.
It would also be advantageous to develop additional compositions that are specifically targeted to other receptors on T lymphocytes. For example, targeting of T lymphocytes would enable therapeutic applications for T-cell-associated diseases and tissue graft rejection. Such T-cell-associated diseases include arthritis, T-cell lymphoma, skin cancers, psoriasis, multiple sclerosis, Type II diabetes mellitus, and diseases resulting from HIV infection.
In view of the foregoing, it will be appreciated that compositions for intracellular delivery of chemical agents to T cells and methods of use thereof would be significant advancements in the art.
It is an object of the present invention to provide compositions for intracellular delivery of selected chemical agents to a specific cell type, i.e. IL-2-receptor-bearing cells.
It is also an object of the invention to provide methods of making and methods of using compositions for delivery of selected chemical agents into IL-2-receptor-bearing cells.
It is another object of the invention to provide compositions and methods for delivering selected chemical agents into IL-2-receptor-bearing cells using water soluble polymers that are inexpensive, FDA-approved, and resistant to development of an antibody response.
It is yet another object of the invention to provide compositions and methods of use thereof for delivery of selected chemical agents into activated T cells.
It is still another object of the invention to providing compositions and methods of use thereof for detecting a disease associated with elevated levels of soluble IL-2 receptor in body fluids, such as serum.
These and other objects are achieved by providing a composition for delivery of a chemical agent into an IL-2-receptor bearing cell, the composition comprising (a) a water-soluble, biocompatible polymer, (b) the chemical agent covalently, releasably coupled to the polymer, and (c) a ligand comprising an IL-2-receptor-binding peptide covalently coupled to the polymer.
In a preferred embodiment of the invention, the composition has a formula selected from the group consisting of Pxe2x80x94[Taxe2x80x94Lxe2x80x94Sxe2x80x94A]c and [Axe2x80x94S]dxe2x80x94Pxe2x80x94[Taxe2x80x94L]c, wherein L is the ligand; A is the chemical agent; S and T are spacers, wherein at least S is biodegradable and S and T can be the same or different; P is the water soluble polymer having functional groups compatible with forming covalent bonds with the ligand; a is 0 or 1; and c and d are integers of at least 1.
Preferably, P is a polyalkylene oxide. Preferred polyalkylene oxides are selected from the group consisting of alpha-substituted polyalkylene oxide derivatives, polyethylene glycol (PEG) homopolymers and derivatives thereof, polypropylene glycol homopolymers and derivatives thereof, alkyl-capped polyethylene oxides, bis-polyethylene oxides, copolymers of poly(alkylene oxides), and block copolymers of poly(alkylene oxides) or activated derivatives thereof. Preferably, the polyalkylene oxide has a molecular weight of about 200 to about 50,000. More preferably, the polyalkylene oxide has a molecular weight of about 2,000 to about 20,000. Most preferably, the polyalkylene oxide has a molecular weight of about 20,000. Especially preferred polyalkylene oxides are polyethylene glycol and polyethylene oxide.
The IL-2-receptor-binding peptide is preferably a member selected from the group consisting of SEQ ID NO: 1 and biologically functional equivalents thereof. More preferably, the IL-2-receptor-binding peptide is a member selected from the group consisting of SEQ ID NO:1 through SEQ ID NO:11, and SEQ ID NO:24 through SEQ ID NO:47.
The chemical agent is preferably selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs.
Preferably, the spacer comprises a peptide. A preferred peptide spacer comprises Gly-Phe-Leu-Gly (SEQ ID NO:21).
In one preferred embodiment, the composition further comprises a carrier selected from the group consisting of other water soluble polymers, liposomes, and particulates. Preferably, such water soluble polymers are selected from the group consisting of dextran, inulin, poly(L-lysine) with modified epsilon amino groups, poly(L-glutamic acid), and N-substituted methacrylamide-containing polymers and copolymers.
A method of delivering a chemical agent in vitro into an IL-2-receptor-bearing cell in a population of cells comprises the steps of:
(a) providing a composition comprising (i) a water-soluble, biocompatible polymer, (ii) the chemical agent covalently, releasably coupled to the polymer, and (iii) a ligand comprising an IL-2-receptor-binding peptide covalently coupled to the polymer; and
(b) contacting the population of cells with an effective amount of the composition under conditions wherein the ligand binds to an IL-2 receptor on the IL-2-receptor-bearing cell and elicits endocytosis of the composition.
A method of delivering a chemical agent into an IL-2-receptor-bearing cell in a warm-blooded animal, comprises the steps of:
(a) providing a composition comprising (i) a water-soluble, biocompatible polymer, (ii) the chemical agent covalently, releasably coupled to the polymer, and (iii) a ligand comprising an IL-2-receptor-binding peptide covalently coupled to the polymer; and
(b) systemically administering to the warm-blooded animal an effective amount of the composition under conditions wherein the ligand contacts and binds to an IL-2 receptor on the IL2-receptor-bearing cell and elicits endocytosis of the composition.
Another aspect of the invention relates to a composition comprising a peptide selected from the group consisting of SEQ ID NO:2 through SEQ ID NO:11 and SEQ ID NO:22 through SEQ ID NO:47 and amides thereof.
A method for detecting a disease associated with elevated levels of soluble interleukin-2 receptor in circulation comprises the steps of:
(a) providing a composition comprising an IL-2-receptor-binding peptide;
(b) mixing the composition with a body fluid to be tested under conditions suitable for binding of the composition to said soluble interleukin-2 receptor in the body fluid to form a complex; and
(c) detecting the complex and determining whether the complex is present at elevated levels as compared to normal individuals.
Diseases that can be detected according to this method include T-cell lymphocytic leukemia, T-cell acute lymphoblastic leukemia, peripheral T-cell lymphoma, Hodgkin""s disease, and non-Hodgkin""s lymphoma. Preferably, the body fluid that is tested is serum. Detection of the complex of peptide and soluble interleukin-2 receptor preferably comprises an enzyme-linked or radio-linked sorbent assay. IL-2 receptor-binding peptides that are suitable for this process include SEQ ID NO:1 through SEQ ID NO:11 and SEQ ID NO:22 through SEQ ID NO:47 and amides thereof. Especially preferred peptides are SEQ ID NO:27 and the amide thereof. As is well known in the art, such an amide is generally formed by reaction of an acid chloride of the peptide with ammonia, resulting in replacement of the xe2x80x94OH group of the C-terminal carboxylic acid with xe2x80x94NH2.